Invasive and deadly metastatic melanoma is associated with excessive exposure to ultraviolet radiation (UVR). UVR induces activating mutations in oncogenes such as BRAF which initiates the development of pigmented lesions (nevi) and melanoma; however it is also believed that UVR promotes melanomagenesis independently of its mutagenic action. Recently, a molecular model of UVR tumor promotion has emerged where melanocyte proliferation seems to be up-regulated by keratinocyte-derived melanocyte growth factors under the control of the transcription factor and key tumor suppressor p53 in response to UVR. Moreover, in melanoma prone mice that carry mutations in Braf (BrafV600E) or CDK4 (CDK4R24C/R24C), increased expression of melanocyte growth factors accelerated the formation of pigmented lesions and promoted melanoma. In line with this, our unique mouse model with constitutively high expression of keratinocyte-specific p53 displayed a propensity to develop nevi and melanoma when subjected to a carcinogenesis protocol. These findings lead us to the hypothesis that keratinocyte p53 releases paracrine melanocyte growth factors that cooperate with oncogenic mutations such as BRAFV600E in melanocytes to promote proliferation of initiated melanocytes and thus melanoma. This novel and important concept provides for the first time a molecular pathway explaining UVR tumor promotion, and extends the role of p53 beyond its canonical tumor suppression to include a tissue- specific function in melanomagenesis. To test this hypothesis we will use intricate human melanocyte and keratinocyte culture systems (Aim 1) and a 3D human skin equivalent xenografted mouse model (Aim 2). In Aim 1, primary human keratinocytes will be UV-irradiated or treated with a specific p53 activating drug, Nutlin- 3a, to generate keratinocyte-conditioned melanocyte growth media. Primary human melanocytes transduced with a control GFP and a GFP-tagged BRAFV600E lentivirus will be grown in the presence or absence of the UVR- or Nutlin 3a-conditioned media. We will monitor the impact of UVR- and p53- conditioned media on the behavior of transduced melanocytes (proliferation, apoptosis, senescence, migration and pigmentation). We expect that paracrine factors in the conditioned media will increase the proliferation and migration potential of BRAF-mutated melanocytes, and decrease their oncogene-induced senescence in culture. Using lentiviral shRNAs, we will identify the most crucial growth factors for melanocytic proliferation and transformation. In Aim 2, human 3D skin equivalent xenografts will be generated that contain the BRAFV600E-GFP and GFP control melanocytes. These grafts will be UV-irradiated or treated with Nutlin-3a to examine the in vivo impact of p53-dependent growth factors on melanocyte behavior as explored in Aim 1. In longer term studies, grafts will be examined for histological melanocytic changes, melanocytic lesions or melanoma. Together, these aims will explore a link that is still not well defined between UVR, BRAF mutations and melanomagenesis which is imperative for our understanding of this deadly tumor. Our findings will demonstrate the involvement of a paracrine p53 signaling in melanoma development and may lead to new strategies for melanoma prevention and treatment.